Clonal analysis of peripheral blood lymphocytes from three patients with advanced neuroblastoma receiving recombinant interleukin-2 and interferon α

In this study we have investigated, at the population and the clonal levels, the immunophenotypes and the non-specific cytotoxic functions of peripheral blood lymphocytes from three stage IV neuroblastoma patients receiving treatment with recombinant interleukin-2 (IL-2) and interferon (IFN). Both IL-2 alone and the combination of IL-2 and IFN caused an in vivo expansion of CD56⁺, CD3^– NK cells most of which expressed the p75 molecule, i.e. the chain of the IL-2 receptor. Peripheral blood mononuclear cells (PBMC), drawn after treatment, displayed an increased NK activity, but no lymphokine-activated killer (LAK) activity. However, the subsequent in vitro culture of PBMC with high-dose IL-2 induced the generation of a potent LAK activity, which was mediated by an expanded population of CD3⁺ CD8⁺ T cells. Finally lymphocytes that had been isolated after cytokine therapy were cloned, in the presence of low-dose phytohemagglutin, immediately or following culture with IL-2. Clones derived from LAK cells expanded in vitro had predominantly a CD3⁺, CD8⁺ immunophenotype, whereas those raised from freshly separated lymphocytes were either CD3⁺, CD4⁺ or CD3⁺, CD8⁺ in equal proportions. Most of the above clones were poorly or not at all cytolytic against NK-sensitive or NK-resistant targets. In contrast, the few NK clones obtained (CD3^–, CD56⁺) lysed all targets with high efficiency.This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro, Milano, Italy to V. P.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Establishment of cytotoxic CD4+ T cell clones from cancer patients treated by local immunotherapy

We have previously reported that the antitumor effect of OK-432, aStreptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (Cancer, 69: 636–642, 1992). In order to elucidate the mechanism of the antitumor effects, we established T cell clones from regional lymph nodes of colorectal cancer patients who received this local immunotherapy. By culture of lymph node lymphocytes, in the presence of IL-2 and OK-432, 4 clones of T cells were established from 4 patients treated by local immunotherapy. These clones had a helper T cell phenotype (CD3⁺, CD4⁺, CD8^–, CD56^–, WT31⁺) and were successfully maintained for several months. The cells strongly expressed CD25 when stimulated with OK-432 and exhibited a high level of cytotoxic activity in part explained by the increased expression of ICAM-1 and LFA-1, and the release of TNF. These results suggest that the CD4⁺ T cells play a role in the antitumor mechanism of local immunotherapy.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

Chinese medicine,Coix seeds increase peripheral cytotoxic T and NK cells

Coix seeds, a Chinese medicine have been used in Japan and reported to be effective in patients with verruca vulgaris and verrucae planae juveniles. We investigated thein vivo effects on lymphocyte subsets in seven healthy volunteers who took six tablets of Coix seeds three times a day (a typical dose) for four weeks. Leukocyte counts and the percentage of total lymphocytes did not change but the percentages of CD3^–CD56⁺ cells and CD16⁺CD57^– cells increased significantly. These results indicate that Coix seeds increase peripheral cytotoxic lymphocytes and may be effective to viral infection through the enhancement of cytotoxic activity.     

Interleukin-2-induced lymphoproliferative responses

Summary Interleukin-2 (IL-2) is capable of both stimulating an in vitro lymphoproliferative response and augmenting non-major-histocompatibility-complex-(MHC)-restricted cytotoxicity. However, there are conflicting reports about the phenotypes of responding cells. In the present studies, we determined phenotypes of Ficoll/Hypaque-separated peripheral blood mononuclear cells stimulated with 50, 100 or 1000 U/ml IL-2; analyses were performed after 1, 3 and 5 weeks. With all concentrations, there was a progressive increase in CD3⁺ cells; after 3–5 weeks more than 90% of the cells reacted with this antibody. However, the proportions of CD4⁺ and CD8⁺ cells proved to be a function of the IL-2 concentration. Cultures containing 50 U/ml or 100 U/ml favored the expansion of the CD4⁺ subset. By contrast, in cultures stimulated with 1000 U/ml, CD8⁺ cells predominated. At baseline, CD8⁺ cells comprised 28±2%; after 3 weeks, this value increased to 51±5%. In addition, the proportion of CD56⁺ (Leu19, NKH-1) cells depended on the amount of IL-2. At 50 U/ml, there was no appreciable change in CD56⁺ cells. However, at 1000 U/ml, CD56⁺ cells increased from 17±1% (day 0) to 39±4% (3 weeks). This increase was primarily due to an expansion of the CD3⁺ CD56⁺ subset (non-NMC restricted cytotoxic T cells). By contrast, natural killer (NK) cells, as measured by the CD16 antibody, steadily declined at all IL-2 concentrations.These studies were supported by a grant from the National Cancer Institute, NIH (RO1 CA24429-15)     

Relationship between CD107a expression and cytotoxic activity

NK cells play important roles in innate immunity against tumors and infections of the host. Studies show that CD107a (LAMP-1) may be a marker for degranulation of NK and activated CD8⁺ T cells. In our study, the relationship between the expression of CD107a, cytokine secretion and cytotoxic activity in CD56⁺ NK, CD8⁺ T cells and lymphocytes has been determined after various stimuli. Effector cells from PBMCs of healthy subjects were isolated and K562 cell line was used as target of cytotoxicity. IL-2 stimulation resulted in a significant increase of CD107a expression in CD56⁺ NK, CD8⁺ T cells and lymphocytes. Increased expression of CD107a after IL-2 stimulation of NK cells was parallel to the increase of cytotoxicity. Our results suggest that CD107a expression may be a sensitive marker for the cytotoxic activity determination.     

Characteristics of the Cells-Producents of Thymic Chemotactic Factor for Bone Marrow Stem Elements

It was shown using complement-dependent cytolysis and monoclonal antibodies against CD4, CD8, and NK1.1 antigens that the cortisone-resistant CD3⁺4^–8^–NK1.1^–-thymocytes spontaneously secreted a chemotactic transmitter inducing the release and directed migration of bone marrow cells. When estimating the general profile of the cytokines of these thymocytes by PCR with revertase, it was demonstrated the cells in question did not express cytokines with colony stimulating activities (SCF, IL-3, or GM-CSF) or cytokines affecting the migration of bone marrow stem elements (IL-2, 4, or 7). In addition, an active expression of gene bcl-2 was detected. Thus, the chemotactic cytokine inducing the release of bone marrow stem elements is a product of the cortisone-resistant long-living CD3⁺4^–8^–NK1.1^–-T-cells of the thymus.     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Analysis of T-cell responses in metastatic melanoma patients vaccinated with dendritic cells pulsed with tumor lysates

In melanoma patients, CD8⁺ cytotoxic T cells have been found recognizing self-proteins of which the expression is restricted to the melanocytic lineage. These melanocyte differentiation antigens are expressed in normal melanocytes as well as in 80–100% of primary and metastatic melanoma. In this report, six HLA-A*0201–subtyped metastatic melanoma patients vaccinated with dendritic cells (DCs) pulsed with autologous tumor lysates and keyhole limpet hemocyanin (KLH) were screened for the presence of CD8⁺ T cells specific for three HLA-A*0201–binding peptides derived from the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase. For this purpose, nonstimulated as well as in vitro peptide-stimulated peripheral blood mononuclear cells (PBMCs) were tested for peptide-specific IFN- release by enzyme-linked immunosorbent spot (ELISpot) assays. Furthermore, expression of the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase in tumor lesions was analyzed by immunohistochemistry before and after vaccination. We also used the ELISpot technique to investigate whether KLH-specific T cells were induced and whether these cells released type 1 (IFN-) and/or type 2 (IL-13) cytokines. Our data show induction of CD8⁺ T cells specific for the melanosomal peptides MART-1/Melan-A_27–35 or tyrosinase_1–9, as well as IFN-–releasing KLH-specific T cells, in two of six vaccinated melanoma patients, but do not support an association between the induction of these T cells and clinical responses.     

Distribution of immune cells and expression of interleukin receptors in ileal Peyer’s patches of calves

Newborn calves lack a mature immune system. The immune system develops with age, but the role of the expression of cytokine receptors in the development of immune cells of Peyer’s patches (PPs) in the intestines of calves in the first 2 months has not yet been elucidated. In this study, the distribution of immune cells and the expression of interleukin (IL) receptors (R) in the ileal PPs of newborn and 2-month-old calves were investigated immunohistochemically with monoclonal antibodies against bovine CD4, CD8, IgM, γδTCR, T19, WC3, WC5, and WC6 antigens. The expression of ILRs was examined with antibodies against CD25 (IL-2Rα), IL-2Rγ, IL-4R, IL-6R, IL-10R, and IL-13R antigens. CD4⁺, CD8⁺, γδTCR⁺, T19⁺, and WC6⁺ cells were found to be more widely distributed in the ileal PPs of 2-month-old calves than in those of newborn calves. Moreover, the expression of CD25 (IL-2Rα), IL-4R, and IL-13R in the ileal PPs of 2-month-old calves was more prominent than that in newborn calves. These data suggest that the immune system of calves at 2 months of age is developed by reactions to foreign antigens and aging.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Altered effector function of peripheral cytotoxic cells in COPD

Background

There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8⁺ T lymphocytes, natural killer (NK; CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results

The proportion of peripheral blood NKT-like (CD56⁺CD3⁺) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56⁺CD3^-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56⁺CD3⁺) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells from smokers and HNS.

Conclusion

In this study, we show that the relative numbers of peripheral blood NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.     

Ozone therapy restores immune dysfunction in refractory idiopathic granulomatous mastitis as a novel potential therapeutic approach

Immunological dysfunction has been suggested to play a major role in the pathogenesis of idiopathic granulomatous mastitis (IGM). We recently showed that ozone therapy was effective in patients with steroid-resistant IGM. This study assessed alterations in intracellular cytokine expression patterns in different T-lymphocyte subsets after ozone therapy in refractory IGM. Peripheral blood T lymphocyte subsets (CD8⁺, CD4⁺, CD4⁺CD25⁺CD127⁻) were analyzed via flow-cytometry for intracellular cytokine expressions IFN-γ, TNF-α, IL-10, and TGF-β before and after completion of 4-month systemic ozone therapy. Ozone therapy significantly increased the CD4⁺IFN-γ⁺ (p = 0.032), CD4⁺TNF-α⁺ (p = 0.028), and the CD8⁺TNF-α⁺ (p = 0.012) T cells. In contrast, significant decreases in CD4⁺ IL-10⁺ (p = 0.047) and CD8⁺IL-10⁺ T cells (p = 0.022) and CD4⁺CD25⁺CD127^−//low Treg cells secreting TGF-β (p = 0.005) were found after ozone therapy. When patients were analyzed according to the response to ozone therapy, patients with a complete remission were more likely to have increased CD3⁻CD16⁺CD56⁺ natural killer cells (p = 0.0027) and decreased CD19⁺ B lymphocytes (p = 0.046) following ozone therapy. Our results suggest that ozone therapy stimulated a T-helper-1 response associated with IFN-γ production and downregulation of TGF-β expression in CD4⁺CD25⁺CD127⁻ Treg cells. These alterations in the immune system following ozone therapy can improve wound healing and restore immune dysfunction in patients with refractory IGM.     

Interleukin-21 Is a Critical Regulator of CD4 and CD8 T Cell Survival during Priming under Interleukin-2 Deprivation Conditions

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8⁺ T cells was reduced, whereas CD4⁺ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8⁺ T cells in comparison to CD4⁺ T cells. However, using transwell co-culture assays we have found that CD4⁺ T cells could rescue the survival of CD8⁺ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8⁺ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4⁺ and CD8⁺ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.     

Regulation of Development of CD56brightCD11c+ NK-like Cells with Helper Function by IL-18

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56^brightCD11c⁺ cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56^brightCD11c⁺ cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56^brightCD11c⁺ cells. CD14⁺ monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56^intCD11c⁺ cells to promote their differentiation to CD56^brightCD11c⁺ cells with helper function. The development of CD56^brightCD11c⁺ cells was suppressed in an IFN-α dependent manner. These results indicate that CD14⁺ monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56^brightCD11c⁺ cells, which in turn modulate the expansion of γδ T cells. CD56^brightCD11c⁺ NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.     

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8⁺ T cells. However, their independent functional contributions in distinct CD8⁺ T cell subsets are not well understood. In this study, CD8⁺ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) CD8⁺ T cells (CD28^Hi), while its expression was significantly lower in effector (CD45RA⁺CD27⁻) CD8⁺ T cells (CD28^Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8⁺ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8⁺ T cells. Co-stimulation of CD28^Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28^Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28^Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8⁺ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8⁺ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8⁺ T cells. However, boosting a recall immune response via memory CD8⁺ T cells or vaccination to stimulate naïve CD8⁺ T cells would require CD28-mediated co-stimulation.